Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human melanoma genomes

This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples. WGS was performed on melanoma cell lines (n = 15) and melanoma fresh frozen tumours (n = 222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined. An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1–2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98–99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection. T...
Source: Pathology - Category: Pathology Tags: Molecular Pathology Source Type: research